Taxanes are diterpene compounds containing a taxane skeleton. For example, taxol is famous as the first identified compound with a taxane ring, which is isolated from the bark of the pacific yew, Taxus brevifolia and is effective for the treatment of leukemia and cancer. Recently, it has been reported that: taxol is capable of curing approximately 30%, 50% and 20% of ovarian, breast and lung cancer patients, respectively, by way of inhibiting depolymerization of microtubules (see: E. K. Rowinsky et al., J. Natl. Cancer Inst., 82:1247-1259 (1990)).
To manufacture taxol, total chemical synthesis, semi-synthesis and extraction methods have been employed in the art.
The total chemical synthesis method, however, has not been practically applied in the industry, since it requires very expensive chemical reagents and the yield is not so high, due to the complicated chemical structure of taxol.
The semi-synthetic method employing precursors such as 10-deacetylbaccatin III, has revealed some drawbacks since it essentially entails complicated and multiple steps of isolating and purifying the taxol precursors from Taxus genus plant and transforming the precursors to taxol.
In this regard, the extraction method by which taxol can be isolated from Taxus genus plants in a direct manner, has prevailed in the art, since it has an advantage of economy. However, the said method has revealed a critical shortcoming that it essentially requires a large amount of yew trees to purify taxol, to give rise to the serious environmental disruption.
Accordingly, the ability of total chemical synthesis, semi-synthesis and extraction method to supply taxol for world-wide chemotherapeutic use is not sufficiently assured; and, needs have continued to exist to develop more improved method for taxol production in light of source of taxol.
Under the circumstances, as a promising alternative to solve said problems, a cell culture method has been proposed in the art.
The cell culture-based process, unlike the prior art, has the following advantages: first, taxol can be produced in a steady manner, regardless of fluctuation of supply of yew plants due to the damage by blight and harmful insects, etc.; secondly, cell cultures can be made in large bioreactors, from which taxol can be massively produced by manipulating culture conditions; thirdly, cell cultures produce a simpler spectrum of compounds compared to prior art methods, considerably simplifying separation and purification; fourthly, a cell culture process can adapt quickly to rapid changes in demand; fifthly, a cell culture process can produce taxol as well as taxane precursors such as baccatin that can be converted to taxol.
A variety of methods for producing taxol by utilizing cultured plant cells have been described in the art:
U.S. Pat. No. 5,019,504 discloses a method for producing taxol and its derivative utilizing cultured cells of Taxus brevifolia. However, the yield of taxol described therein is 1-3 mg/L, which is insufficient for industrial application. Besides, the production of taxol by the cell culture is unstable, and even when a primary cell of high productivity can be obtained by selection, it is difficult to keep its content by subculturing (see: E. R. M. Wickremesine et al., World Congress on Cell and Tissue Culture (1992)).
U.S. Pat. No. 5,015,744 teaches a semi-synthetic method from baccatain III, which is a precursor in biosynthesis of taxol. By the use of the plant tissue culture, a raw material for the semi-synthetic process such as baccatin III can be produced, thus the plant tissue culture can also be utilized for taxol production by the above-mentioned semi-synthetic process.
WO 93/17121 offers a method for taxol production by cell culture of Taxus genus plant while changing composition of medium, growth rate, and production rate, etc. In case of Taxus chinensis, 24.1 mg/L of taxol can be obtained in 18 days of culture and biomass doubles every 2.5 days.
U.S. Pat. No. 5,407,816 describes that Taxus chinensis cells are inoculated on a nutrient medium to form a suspension culture and subcultured to a fresh medium to form a producing culture, from which taxol and taxanes can be obtained in a yield of 153 mg/L. The said method was considerably improved in light of taxol productivity, however, it has been proven to be less satisfactory in the sense that it essentially requires so many nutrient media whose compositions are so complicated, and the high productivity can be realized under a rather limited growth condition.
On the other hand, a method for taxol production comprising a step of adding stimulators into culture medium in the course of culturing Taxus genus plant cell to increase productivity of taxol has been suggested in the art. For example, U.S. Pat. No. 5,637,484 discloses an increase of taxol productivity by the addition of jasmonate and Ag-containing compounds into culture medium, where a relatively high concentration (i.e., .about.1000 .mu.M) of complex ion containing Ag is essentially required. However, high level of the Ag-containing compound in the medium plays as an obstacle to normal growth of Taxus genus plant cell, which finally causes low productivity of taxol.
Therefore, there are strong reasons for exploring and developing alternative means for taxol production, which is improved in the senses of productivity and simplicity which are key criteria of a useful method utilized in industrial scale.